Hepatitis C virus (HCV) exists in nature as a genetically diverse quasispecies. Globally, the virus is represented by seven major genotypes (gt 1-7) that differ by as much as 30-35% in nucleotide sequence. A critical obstacle to progress in understanding HCV biology and virus-host interactions leading to virus persistence or eradication has been the extraordinary challenges faced in generating full-length molecular viral clones that replicate efficiently in vitro and in vivo. To date, there is only a single gt 2a clone pJFH1, and chimeric derivatives thereof, that replicate efficiently in vitro and in vivo. This has also hindered the development of effective therapeutic agents and treatment strategies for eradication of different viral genotypes. The present application aims to overcome this scientific roadblock by employing a novel experimental strategy developed in our laboratory based on single-genome sequencing (SGS), to identify and clone actual transmitted/founder (T/F) full-length HCV genomes responsible for productive clinical infection in humans. Our hypothesis is that T/F genomes, which by definition contain all of the genetic elements that are necessary and sufficient for productive virus infection and replication in humans, will replicate in tissue cultue and in small animal models, recapitulating the biology of HCV, and allowing for a detailed molecular assessment of the sensitivity of these viruses to interferons (IFNs), direct-acting antiviral agents (DAA) and other novel therapeutic interventions. Our experimental approach is adapted from our recent work with HIV-1 where we demonstrated that full-length, replication-competent, transmitted HIV-1 genomes could be identified, cloned, expressed and analyzed for biological properties that are essential for transmission and productive infection (J Exp Med 206:1273, 2009; PLoS Pathog 6:e1000890, 2010; PLoS Pathog 8:e1002686, 2012). More recently, we demonstrated the feasibility of this approach for identifying T/F HCV genomes by analyzing subgenomic (5' half-genome) plasma vRNA sequences in 17 acutely infected human subjects (Li et al., PLoS Pathog, in press). In the current project, specific aims are to: (i) molecularly identify by SGS full-length T/F HCV genomes corresponding to gt 1a, 1b, 2b and 3a from acutely-infected human subjects (R21); (ii) chemically synthesize and clone these genomes and launch their replication in human liver chimeric mice (R21); (iii) analyze the expressed T/F genomes in a hierarchy of in vitro biological assays for replication competence, virion production, infectivity, and sensitivity to type I, II and III IFNs and DAAs (R33); and (iv) expand the number and genotypic representation of HCV T/F clones to include gt 1a, 1b, 2b, 3a and 4a (n=3 for each), identify genotype-associated determinants of IFN and DAA sensitivity, and evaluate in tissue culture and small animal models strategies for achieving synergy between therapeutic modalities that lead to virus eradication (R33). The proposed work will provide new insights into the molecular biology of HCV replication and persistence and facilitate strategies for eradicating globally circulating HCV genotypes.